Interactions Between Synthetic Vanilloids and the Endogenous Cannabinoid System

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provided by Elsevier - Publisher Connector Interactions between synthetic vanilloids and the endogenous cannabinoid system

Vincenzo Di Marzo1;a;*, Tiziana Bisognoa, Dominique Melcka, Ruth Rossb, Heather Brockieb, Lesley Stevensonb, Roger Pertweeb, Luciano De Petrocellisc

aIstituto per la Chimica di Molecole di Interesse Biologico, CNR, Via Toiano 6, 80072, Arco Felice, Napoli, Italy bDepartment of Biomedical Sciences, University of Aberdeen, Aberdeen, UK cIstituto di Cibernetica, CNR, Via Toiano 6, 80072, Arco Felice, Napoli, Italy

Received 14 July 1998; received in revised form 7 September 1998

of these receptors was recently cloned and shown to mediate Abstract The chemical similarity between some synthetic agonists of vanilloid receptors, such as olvanil (N-vanillyl-cis-9- capsaicin e¡ects in dorsal root ganglia [2]. The presence of at octadecenoamide), and the èndocannabinoid' anandamide (ara- least one additional vanilloid receptor is suggested by studies chidonoyl-ethanolamide, AEA), suggests possible interactions carried out with resiniferatoxin, a phorbol derivative, the pat- between the cannabinoid and vanilloid signalling systems. Here tern of whose pharmacological actions di¡ers from that of we report that olvanil is a stable and potent inhibitor of AEA capsaicin [3]. A synthetic vanilloid derivative and anti-in£am- facilitated transport into rat basophilic leukemia (RBL-2H3) matory/analgesic compound, N-(3-methoxy-4-hydroxy-ben- cells. Olvanil blocked both the uptake and the hydrolysis of zyl)-cis-9-octadecenoamide (olvanil, [4,5], Fig. 1), was also 14 [ C]AEA by intact RBL-2H3 cells (IC50 =9 WM), while suggested to bind to a special subtype of vanilloid receptors capsaicin and pseudocapsaicin (N-vanillyl-nonanamide) were based on its non-pungency, lower toxicity and slower activa- much less active. Olvanil was more potent than previously tion kinetics of Ca2 inward currents as compared to capsai- reported inhibitors of AEA facilitated transport, i.e. phloretin cin [5,6]. It is not known whether olvanil binds to the recently (IC50 =80 WM), AM404 (12.9% inhibition at 10 WM) or oleoylethanolamide (27.5% inhibition at 10 WM). Olvanil was a cloned VR1 vanilloid receptor [2]. poor inhibitor of [14C]AEA hydrolysis by RBL-2H3 and The pharmacology and biochemistry of cannabinoid recep- N18TG2 cell membranes, suggesting that the inhibitory effect tors are better known than those of vanilloid receptors (for a on [14C]AEA breakdown observed in intact cells was due to recent review see [7]). Two di¡erent receptor subtypes, named inhibition of [14C]AEA uptake. Olvanil was stable to enzymatic CB1 and CB2, have been identi¢ed and cloned from mamma- hydrolysis, and (i) displaced the binding of high affinity lian tissues [7], and endogenous ligands for these proteins have cannabinoid receptor ligands to membrane preparations from been found. The best known èndocannabinoid' is anand- N18TG2 cells and guinea pig forebrain (Ki = 1.64^7.08 WM), but amide (arachidonoylethanolamide, AEA, [8]), whose human not from cells expressing the CB2 cannabinoid receptor subtype; and rat tissue distribution, biosynthetic and catabolic path- (ii) inhibited forskolin-induced cAMP formation in intact ways, and pharmacological actions have been thoroughly N18TG2 cells (IC50 = 1.60 WM), this effect being reversed by the selective CB1 antagonist SR141716A. Pseudocapsaicin, but studied (recently reviewed in [9]). It has been proposed that not capsaicin, also selectively bound to CB1 receptor-containing the action of AEA is terminated by facilitated transport into membranes. These data suggest that some of the analgesic cells followed by enzymatic hydrolysis of the amide bond. actions of olvanil may be due to its interactions with the Evidence for a membrane `carrier' protein selective for AEA endogenous cannabinoid system, and may lead to the design of a has been provided for rat central neurons [10,11] and rat novel class of cannabimimetics with potential therapeutic cerebellar granule cells [12] ^ which selectively express CB1 applications as analgesics. receptors [7] ^ and rat basophilic leukemia (RBL-2H3) cells z 1998 Federation of European Biochemical Societies. [13] ^ which contain CB2 receptors [7,16]. AEA facilitated transport is inhibited by alkylating agents [13], phloretin Key words: Vanilloid receptor; Cannabinoid receptor; Endocannabinoid; Anandamide; Capsaicin; In£ammation; and oleoylethanolamide [12], and some aromatic derivatives Antinociception; Mast cell of AEA such as N-benzyl-arachidonamide [12] and N-(4-hy- droxyphenyl)-arachidonamide (AM404) [11]. Once inside cells, AEA is hydrolyzed to arachidonate and ethanolamine via the action of `fatty acid amide hydrolase' (FAAH), an 1. Introduction enzyme which recognizes as substrates several long chain fatty acid amides and esters [14,15], including a potent anti-in£am- Evidence exists for the presence, in sensory neurons and matory congener of AEA, palmitoylethanolamide (PEA, some peripheral tissues, of distinct types of vanilloid receptors [13,16]). that are activated by the natural component of chili peppers, Starting from the chemical similarity between long chain capsaicin (N-(3-methoxy-4-hydroxy-benzyl)-8-methyl-6-trans- capsaicin analogues and AEA or, particularly, aromatic inhib- nonenamide or N-vanillyl-8-methyl-6-trans-nonenamide, Fig. itors of AEA facilitated transport [11,12], we have examined 1), and antagonized by capsazepine (for a review [1]). One whether capsaicin-like compounds could interact with the best characterized proteins of the èndogenous cannabinoid sys- *Corresponding author. Fax: (39) (81) 8041770. tem', i.e. the AEA `carrier', FAAH and the two cannabinoid E-mail: [email protected] receptor subtypes. We report that olvanil is a potent inhibitor 1Affiliated with the National Institute for the Chemistry of Biological of AEA facilitated transport into RBL-2H3 cells, and both Systems, CNR. olvanil and another capsaicin congener, N-vanillyl-nonan-

FEBS 20935 8-10-98 450 V. Di Marzo et al./FEBS Letters 436 (1998) 449^454 amide (pseudocapsaicin), but not capsaicin, weakly bind to binding was calculated by using either 1 WM unlabelled CP 55 940, CB1, but not CB2, cannabinoid receptors. or 10 WM unlabelled SR 141716A (kindly donated by Sano¢ Re- cherche, Montpellier) or HU-210 (kindly donated by Prof. R. Me- choulam, The Hebrew University of Jerusalem). The concentration 2. Materials and methods of unlabelled ligand that produced a 50% displacement of the labelled ligands from speci¢c binding sites was calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Ki values were Mouse neuroblastoma N18TG2, mouse J774 macrophages and calculated by means of the Cheng-Pruso¡ equation from the K val- 14 d RBL-2H3 cells were cultured as described previously [9,13]. [ C]AEA ues for each radiolabelled ligand in each of the membrane systems 14 and [ C]PEA (5 mCi/mmol) were synthesized as described previously used. Cyclic AMP (cAMP) assays were performed in the Naples lab- 14 [8] starting from [ C]ethanolamine and the corresponding fatty acyl oratory on intact con£uent N18TG2 cells plated in six-well dishes and chlorides. Anandamide and oleoylethanolamide were synthesized like- stimulated for 10 min at 37³C with 1 WM forskolin in 400 Wl of serum- wise from ethanolamine and arachidonoyl- or oleoyl-chlorides. free Dulbecco's modi¢ed Eagle's Medium containing 20 mM HEPES, 3 [ H]SR 141716A (55 Ci/mmol) was purchased from Amersham, 0.1 mg/ml BSA, 0.1 mM 1-methyl-3-isobutylxanthine (Sigma) and 3 3 whereas [ H]CP 55 940 (164 Ci/mmol) and [ H]WIN 55 212-2 (43 Ci/m- either vehicle (ethanol) or anandamide, olvanil and olvanil plus SR mol) were purchased from NEN. Phloretin, capsaicin and pseudocap- 141716A at the concentrations shown in Fig. 3. After the incubation saicin were purchased from Sigma, forskolin from Fluka, and AM404 cells were extracted, and cAMP levels determined by means of a from Cayman Chemical. Olvanil was synthesized in the Naples labo- cAMP assay kit (Amersham), as advised by the manufacturer. ratory by reacting 30 mg of 3-methoxy-4-hydroxy-benzylamine with an excess of oleoylchloride at 4³C for 30 min. The mixture was then brought to dryness under a £ow of N2 and puri¢ed by normal phase 3. Results and discussion high pressure liquid chromatography (NP-HPLC) carried out with a semi-preparative column (Spherisorb S5W) eluted with a 40-min lin- In a previous study [13] we showed that, in RBL-2H3 cells, ear gradient from 90% to 80% of n-hexane in 2-propanol (£ow rate 2 ml/min). Under these conditions olvanil was eluted after 27 min. distinct uptake mechanisms exist for AEA and its anti-in£am- Electron-impact mass spectrometric analysis, carried out on an HP- matory congener PEA [16]. The di¡usion of both acylethanol- 5989B quadrupole mass analyzer equipped with an electron impact amides into intact RBL-2H3 cells was maximal after 20^30- source operating at 70 eV and 250³C, con¢rmed the chemical struc- min incubations, saturable, inhibited by N-ethylmaleimide ture of the compound (m/z = 417, molecular ion; m/z = 294, loss of a 3-methoxy-4-hydroxy-phenyl group; m/z = 137 and 281, cleavage of and PMSF, and greatly reduced at 0^4³C. However, the two the CH2-NH bond). Experiments with intact cells were carried out compounds did not compete for each other's uptake. RBL- as described previously [13] in six-well dishes (about 1.0U106 cells/ 2H3 cells were also found to express an `AEA amidohydro- well). Cells were incubated at 37³C with 0.5 ml of serum-free media lase' activity [13] which was later identi¢ed as FAAH [17]. In 14 14 containing 20 000 cpm (4 WM) of either [ C]AEA or [ C]PEA agreement with the presence of distinct transporter proteins for 30 min with the substances shown in Figs. 1 and 2. After having washed the cells three times with 3 ml of 0.2% bovine serum for the two acylethanolamides in RBL-2H3 cells, here we albumin (BSA)-containing medium, [14C]AEA or [14C]PEA and found that olvanil inhibits the facilitated di¡usion of [14C]ethanolamine in media and cells were quanti¢ed as described [14C]AEA, but not [14C]PEA, thereby signi¢cantly increasing [13]. Some experiments were also performed in the presence of 50 the amount of extracellular AEA potentially available for WM p-hydroxymercuribenzoate. [14C]AEA and [14C]PEA hydrolysis by cell-free fractions was studied by using 10 000Ug pellets from cannabinoid receptor activation (Fig. 2A and data not RBL-2H3 and N18TG2 cell homogenates prepared as described shown). The inhibitory e¡ect of olvanil (estimated [13]. Aliquots of 40^80 Wg total proteins were incubated in 0.5 ml of 50 mM Tris-HCl, pH 9, with 40 000 cpm of either [14C]AEA or [14C]PEA for 30 min at 37³C. [14C]Ethanolamine produced from the enzymatic hydrolysis was determined as described elsewhere [13]. Ex- periments on the hydrolysis of olvanil (0.2 mg) by N18TG2 or RBL- 2H3 cell membrane preparations were carried out with 30^300 Wg total proteins suspended in 0.5 ml Tris-HCl, 50 mM, pH 9.0, and incubated for 30 min at 37³C. Aliquots of the membrane preparations were incubated in parallel with [14C]AEA to con¢rm the presence of FAAH activity. After the incubation, lipids were extracted and loaded on analytical thin layer chromatography plates in comparison with synthetic standards of olvanil and oleic acid. Plates were developed with chloroform/ methanol/NH3 85:15:1 by vol. and the formation of oleic acid (Rf = 0.25) monitored by exposing the plate to iodine va- pors. All the experiments on the e¡ects of olvanil, capsaicin and pseudocapsaicin on AEA and PEA inactivation were performed in the Naples laboratory. Binding assays were carried out with guinea- pig forebrain (GPF) membranes or with membranes from CHO cells stably transfected with CB2 receptors (CHO-CB2 cells) [18] (in the Aberdeen laboratory), or with membranes from N18TG2 and RBL- 2H3 cells (in the Naples laboratory). Forebrains from adult male Dunkin Hartley guinea-pigs were suspended in 50 mM Tris bu¡er (pH 7.4) and 0.32 M sucrose and homogenized with an Ultra-Turrax homogenizer. The homogenates were diluted with 50 mM Tris bu¡er and centrifuged at 100 000Ug for 1 h thus yielding the membranes for the binding assays. CHO-CB2, N18TG2 and RBL-2H3 cell membranes were prepared as described in [18,19] and [16], respectively. Binding assays were carried out by using the ¢ltration procedures described previously [16,18,19] with slight modi¢cations consisting in the presence of phenylmethylsulfonyl£uoride (PMSF, Sigma, 0.1 mM) in the respective binding bu¡ers, and the use of di¡erent amounts of total membrane proteins and radiolabelled ligands ([3H]CP 55 940 for GPF and CHO-CB2 membranes, [3H]SR 141716A for N18TG2 mem- Fig. 1. Chemical structures of capsaicin, pseudocapsaicin and branes, and [3H]WIN 55 212-2 for RBL-2H3 membranes). Speci¢c olvanil.

FEBS 20935 8-10-98 V. Di Marzo et al./FEBS Letters 436 (1998) 449^454 451

Fig. 2. E¡ect of olvanil and other inhibitors of AEA facilitated transport on [14C]AEA inactivation by RBL-2H3 cells. A: Dose-dependent inhibition by olvanil of [14C]AEA facilitated di¡usion into and hydrolysis by intact cells. Data are expressed as radioactivity associated with AEA in the incubation media or inside cells, or with ethanolamine produced by AEA hydrolysis, after a 30-min incubation at 37³C, and are means þ S.D. of three separate experiments carried out in duplicate. No signi¢cant e¡ect of olvanil on [14C]PEA facilitated di¡usion and hydrolysis in intact cells was observed. The e¡ect of olvanil in the presence of 50 WM p-hydroxymercuribenzoate is shown in the inset. B: Dose-dependent e¡ect of phloretin, AM404 and oleoylethanolamide on [14C]AEA facilitated di¡usion into intact cells. Data are expressed as radioactivity associated with AEA inside cells after a 30-min incubation at 37³C, and are means þ S.D. of three separate experiments carried out in duplicate. C: Dose-dependent inhibition by olvanil of [14C]AEA and [14C]PEA hydrolysis by RBL-2H3 membrane preparations (10 000Ug pellets). Data are expressed as the radioactivity associated with ethanolamine produced by AEA or PEA hydrolysis after a 30-min incubation at 37³C, and are means of three separate experiments carried out in duplicate. S.D. bars are not shown for the sake of clarity and were never higher than 5%. C

14 IC50 = 9 þ 2 WM, n = 3) on [ C]AEA di¡used into cells after 30-min incubations at 37³C was more potent than that observed here with previously described [11,12] blockers of AEA facilitated di¡usion such as phloretin (estimated IC50 = 80 WM), oleoylethanolamide (27.5% inhibition at 10 WM) or AM404 (12.9% inhibition at 10 WM) (Fig. 2B). The data ob- tained with the former two compounds are in good agreement with those previously reported for the inhibition of AEA dif- fusion into rat cerebellar granule cells [12], also in view of the fact that the concentration of radiolabelled AEA used in the previous study (0.3 nM) was much lower than that used here (4 WM). The high concentration of the radiolabelled ligand used in the present study may indeed explain why AM404 was much less e¡ective against [14C]AEA uptake by RBL- 2H3 cells than previously described for cortical astrocytes and neurons (IC50 = 1^5 WM, [11]). We thought that this could have been due also to the longer incubation time used here as compared to the previous study [11], and, therefore, performed experiments looking at the e¡ect of inhibitors on [14C]AEA di¡used into cells after 15-min incubations. This resulted in a slightly increased potency for oleoylethanolamide (32.1% inhibition at 10 WM) but not AM404 (14.5% inhibition at 10 WM) (data not shown). With 30-min incubations, olvanil also inhibited [14C]AEA hydrolysis by intact RBL-2H3 cells with an estimated IC50 of 9 WM (Fig. 2A). Since the vanilloid compound inhibited [14C]AEA hydrolysis by RBL-2H3 membrane preparations at higher concentrations (IC50 = 32 þ 1 WM, n = 3, Fig. 2C), it is possible that the inhibitory e¡ect observed in intact cells was due to inhibition of AEA facilitated di¡u- sion, which precedes enzymatic hydrolysis (as shown in [10], 14 the t1=2 values for AEA uptake and hydrolysis by rat cortical greatly a¡ecting [ C]AEA uptake (respectively, 42.2 þ 5.3 neurons are 2.5 and 6 min, respectively; moreover, the sub- and 115.5 þ 1.1% of control, see also [13]). We found that, cellular distribution of FAAH, described in [29], seems to rule under these conditions, olvanil still inhibited [14C]AEA accu- out that anandamide is hydrolyzed by intact cells prior to its mulation (IC50 = 35 þ 2 WM, n = 3, Fig. 2A, inset) with little internalization). Olvanil also inhibited [14C]AEA hydrolysis by e¡ect on residual [14C]AEA hydrolysis (41.8 þ 2.3% inhibition N18TG2 membrane preparations ^ where FAAH expression at 100 WM), thus showing that the e¡ect of the vanilloid on has been shown [17] ^ but again only at high concentrations AEA uptake is not due to inhibition of AEA hydrolysis. (IC50 = 48 þ 2 WM). However, in order to isolate the e¡ect of Three further sets of data are in agreement with this conclu- olvanil on the AEA carrier from its weak e¡ect on FAAH, we sion: (i) olvanil also inhibited [14C]AEA accumulation in J774 studied the uptake of [14C]AEA by RBL-2H3 cells in the macrophages ^ where negligible FAAH activity is expressed presence of p-hydroxymercuribenzoate at a concentration [13,17] ^ with an estimated IC50 = 27 þ 9 WM (n = 3); (ii) ole- (50 WM) found to counteract [14C]AEA hydrolysis without amide, a competitive inhibitor of [14C]AEA hydrolysis and a

FEBS 20935 8-10-98 452 V. Di Marzo et al./FEBS Letters 436 (1998) 449^454

activation in intact cells (IC50 s 100 WM) and only slightly a¡ected [14C]AEA hydrolysis by membrane preparations (IC50 = 83 WM and 39 WM, respectively). We next carried out a series of cannabinoid receptor binding experiments with olvanil, capsaicin and pseudocapsaicin. We used membrane preparations from guinea pig forebrain (GPF) and mouse neuroblastoma N18TG2 cells, which selectively express the CB1 receptor subtype [18,19], as well as membranes from CHO cells transfected with CB2 receptor cDNA (CHO-CB2 cells). The high a¤nity ligand [3H]CP 55 940, which binds to both cannabinoid receptor subtypes, was used for GPF and CHO-CB2 cell membranes, whereas the selective CB1 antagonist [3H]SR 141716A was used for N18TG2 cell membranes [19]. We found that olvanil could displace both radiolabelled ligands from CB1 receptor-containing membranes, although only at WM concentrations, but not [3H]CP 55 940 from CHO-CB2 cell membranes (Table 1). Furthermore, olvanil was almost inactive in displacing [3H]WIN 55 212-2 from RBL-2H3 cell membranes, which Fig. 3. Dose-dependent inhibition by olvanil and anandamide of also selectively express CB2 receptors [16]. The Ki values ob- forskolin-induced cAMP formation in N18TG2 cells. The e¡ect of 3 0.5 WM SR 141716A on olvanil inhibitory action is also shown. tained for olvanil for the displacement of [ H]CP 55 940 from Data are expressed as percent of cAMP levels in cells stimulated GPF membranes varied between 4.54 and 11.98 WM, with an with 1 WM forskolin (66.6 þ 14.1 pmols/well), and are means average value of 7.08 WM. AEA in the same assay exhibited a þ S.E.M. of three separate experiments. cAMP levels in unstimu- Ki value of 0.48 WM. The Ki for olvanil displacement of lated cells were 7.7 þ 3.3 pmols/well. [3H]SR 141716A from N18TG2 cell membranes was signi¢- cantly lower (1.64 þ 0.36 WM, n = 3) and similar to that found for AEA (1.91 þ 0.31 WM, n = 3). One reason for these discrep- FAAH substrate [9,14,15,17], did not a¡ect [14C]AEA uptake ancies may be the use of di¡erent experimental procedures, by RBL-2H3 cells (data not shown); and (iii) olvanil did not membrane preparations and radioligands in these experiments 14 counteract [ C]PEA di¡usion into RBL-2H3 cells even (see Section 2). Indeed, £uctuations in the Ki values reported though it inhibited [14C]PEA hydrolysis by membrane prepa- for anandamide (from low-middle to high nM concentrations) rations as weakly as it did with [14C]AEA hydrolysis when using di¡erent procedures have been pointed out [7], (IC50 = 49 WM, Fig. 2C). We also found that olvanil is stable and di¡erences of more than 50-fold exist between some of to FAAH-catalyzed hydrolysis since, under conditions leading the reported Ki values of CP 55 940 for the displacement of to e¤cient [14C]AEA hydrolysis, no oleic acid formation was [3H]CP 55 940 from CB1 receptors (see Table 2 in [7]). An- observed from incubation of the vanilloid (0.4 mg/ml) with other possibility, i.e. that olvanil is more potent in displacing either RBL-2H3 or N18TG2 cell membranes (data not [3H]SR 141716A than [3H]CP 55 940 because olvanil and SR shown). 141716A behave as inverse agonists, is ruled out by our data. We wanted to assess whether capsaicin and its analogue In fact, although SR 141716A under certain conditions may pseudocapsaicin also inhibited AEA facilitated transport. be an inverse agonist ([7,9] and references cited therein), we The two compounds were found to reduce the uptake and found olvanil to behave as a cannabinoid receptor agonist. the hydrolysis of [14C]AEA by intact RBL-2H3 cells only at We measured the e¡ect on forskolin-induced cAMP forma- high WM concentrations, capsaicin being the most e¡ective tion ^ an intracellular event whose inhibition is coupled to one on the uptake (estimated IC50 = 46 WM, with IC50 s 100 CB1 receptor activation [20] ^ in intact N18TG2 cells, which WM for the hydrolysis; the IC50 values for pseudocapsaicin have been often utilized to study the e¡ect of cannabimimetic were 83 and 60 WM for hydrolysis and uptake, respectively). compounds on adenylate cyclase [20]. As shown in Fig. 3, The two compounds were not e¡ective against [14C]PEA in- olvanil behaved as an agonist in this assay, and exhibited an

Table 1 Ki values (WM) for olvanil, pseudocapsaicin and anandamide binding to membrane preparations from various sources using di¡erent cannabinoid receptor ligands CB1 CB2 GPF+[3H]CP 55 940 N18TG2+[3H]SR 141716A CHO-CB2+[3H]CP 55 940 RBL-2H3+[3H]WIN 55 212-2 Olvanil 7.08 þ 1.41 1.64 þ 0.36 s 20 s 15 Pseudocapsaicin 6.16 þ 0.13 1.20 þ 0.28 s 20 N.D. Anandamide 0.48 þ 0.04 1.91 þ 0.31 0.371 þ 0.102 [18] 0.03 [16] Concentrations in the range 0.1^10 WM were tested. Data are means þ S.E.M. of at least three separate experiments. For olvanil displacement of 3 3 [ H]CP 55 940 from CHO-CB2 cell membranes and of [ H]WIN 55 212-2 from RBL-2H3 cell membranes, Ki values higher than 20 and 15 WM can be predicted from the observation that the compound exhibited, respectively, 0 and 6.7 þ 2.3% displacement at 1 WM and 19.5 þ 1.5% and 32.0 þ 2.1% displacement at 10 WM. The same applies to pseudocapsaicin (15.8 þ 2.8% displacement at 10 WM for CHO-CB2 cell membranes). The Ki values for anandamide in these two cell membrane preparations, calculated by using a methodology similar to the ones described here, have been already reported [16,18]. N.D. = not determined.

FEBS 20935 8-10-98 V. Di Marzo et al./FEBS Letters 436 (1998) 449^454 453

IC50 value of 1.60 WM similar to the Ki value for the displace- from rat or mouse lumbar spinal cord [23], it is unlikely that ment of [3H]SR 141716A observed in membranes from the the endocannabinoid interacts with vanilloid receptors. In same cells. Olvanil was more potent than AEA (IC50 = 3.20 conclusion, apart from suggesting the necessity of a general WM), probably due to its major stability to enzymatic degra- re-evaluation of the pharmacology and biochemistry of long dation (see above). Furthermore, olvanil inhibition of forsko- chain capsaicin analogues (reviewed in [28]), the data de- lin-induced cAMP formation was reversed by 0.5 WM SR scribed in this paper support a case for interactions between 141716A, thus proving that this e¡ect was mediated by CB1 some synthetic vanilloids and the cannabinoid system and receptors. Clearly, further studies are still needed in order to prompt further studies in this direction. Furthermore, the characterize the CB1-binding properties of olvanil more fully. ¢nding of the weak cannabimimetic actions of olvanil and These studies may also indicate which of the structural fea- pseudocapsaicin may open the way to the design of a novel tures of vanilloids need to be modi¢ed in order to improve class of analgesics with a multiple mechanisms of action. their weak CB1-binding activity. Interestingly, we found that also pseudocapsaicin displaced cannabinoid ligands from Acknowledgements: This work was supported by grants from the Hu- CB1, but not CB2, receptor-containing membrane prepara- man Frontier Science Program Organization (RG26/95 to V.D.M.) and the Wellcome Trust (047980 to R.P. and R.R.). The authors tions (Ki = 6.16 WM for GPF membranes and 1.20 WM for are grateful to Sano¢ Recherche, Montpellier, France, for the gift N18TG2 cell membranes, Table 1), whereas capsaicin caused of SR 141716A. no displacement at 10 WM (data not shown). This ¢nding is in agreement with the widely accepted concept that a linear, References saturated aliphatic chain of at least ¢ve carbon atoms (Fig. 1) must be present in the molecule to allow the interaction [1] Dray, A. (1992) Biochem. Pharmacol. 44, 611^615. with CB1 receptors [30]. [2] Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., In summary, we have shown that the long chain analogue Levine, J.D. and Julius, D. (1997) Nature 389, 816^824. of capsaicin, olvanil, can e¤ciently interact with at least one [3] Liu, L., Szallasi, A. and Simon, S.A. (1998) Neuroscience 84, 569^581. of the four proteins of the `endogenous cannabinoid system' [4] Sietsema, W.K., Berman, E.F., Farmer, R.W. and Maddin, C.S. studied here. Olvanil (i) inhibits AEA facilitated transport (1988) Life Sci. 43, 1385^1391. into RBL-2H3 cells; (ii) is a weak agonist at CB1, but not [5] Liu, L., Lo, Y., Chen, I. and Simon, S.A. (1997) J. Neurosci. 17, CB2, receptors; and (iii) very weakly inhibits AEA hydrolysis 4101^4111. by FAAH-containing membrane preparations. Except for a [6] Wardle, K.A., Ranson, J. and Sanger, G.J. (1997) Br. J. Phar- macol. 121, 1012^1016. weak inhibition of AEA uptake, capsaicin does not exhibit [7] Pertwee, R.G. (1997) Pharmacol. Ther. 74, 129^180. similar properties, whereas pseudocapsaicin is inactive on [8] Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, the AEA transporter but weakly binds to CB1 receptors. L.A., Gri¤n, G., Gibson, D., Mandelbaum, A., Etinger, A. and Both AEA and olvanil were shown previously to exert potent Mechoulam, R. (1992) Science 258, 1946^1949. [9] Di Marzo, V. (1998) Biochim. Biophys. Acta 1392, 153^175. vasodilatory, analgesic and anti-in£ammatory actions [4^ [10] Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., 6,21^24], although probably with di¡erent mechanisms of ac- Schwartz, J.-C. and Piomelli, D. (1994) Nature 372, 686^691. tion. While such e¡ects of AEA seem to be mediated by can- [11] Beltramo, M., Stella, N., Calignano, A., Lin, S.Y., Makriyannis, nabinoid receptors located on sensory/sympathetic ¢bers, A. and Piomelli, D. (1997) Science 277, 1094^1097. smooth muscle, endothelial cells and leukocytes [7,21^23], ol- [12] Hillard, C.J., Edgemond, W.S., Jarrahian, A. and Campbell, W.B. (1997) J. Neurochem. 68, 631^638. vanil actions are probably due to interaction with ^ and sub- [13] Bisogno, T., Maurelli, S., Melck, D., De Petrocellis, L. and Di sequent desensitization of ^ selective polymodal sensory noci- Marzo, V. (1997) J. Biol. Chem. 272, 3315^3323. ceptors, the vanilloid receptors [5]. While AEA has been [14] Cravatt, B.F., Giang, D.K., May¢eld, S.P., Boger, D.L., Lerner, recently shown to inhibit the release of calcitonin gene-related R.A. and Gilula, N.B. (1996) Nature 384, 83^87. 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